A standard protocol for intracellular staining usually separate the fixation and permeabilization part.
Can I do the fixation and permeabilization at one time by incubating cells with 3.7% formaldehyde with 0.1% Triton-X in ice for 30mins?
I include 0.2% TX-100 in my blocking buffer/immunofluorescence diluent. It seems to work well, no permeabilization necessary (as you permeabilize during the blocking/staining).
My immunofluorescence/blocking buffer recipe is PBS with 2% normal goat serum, 0.2% triton x-100 and 0.05% sodium azide.
Combining fixation and permeabilization should work, as it's done with much gentler detergents and formaldehyde for FACS (see BD Cytofix/Cytoperm).
Hi Ki Siang Lim
Mixing of formaldehyde with permeabilizing agent is in fact unnecessary. Formaldehyde is quite a small molecule and is able to crosslink proteins on its own (fixation step).
It is for the antibody binding step, permeabilization is required. In my experience diluting the antibody in a mixture of 50% blocking solution and 50% 0.1% Triton-x-100 (in PBS) provides good enough blocking and permeabilization effect. It gives very good staining results.
Good Luck!
U may fix the cells on cover slip using 4% para formaldehyde , for 15 mins @ room temp, followed by washing and permeablization using 0.05-0.1% tritonx 100 or what u can do -if you want to stain a nuclear antigen u can directly use acetone:methanol(1:1) mixture for fixing as well as permeablize(@ 0 degree for 10 mins), no seperate step for permeablization is required or you can use methanol for both the purposes.
all d best.
For the regular action of fomaldeyhe the pH of the solution has great impact (7,0-7,6). It would be interesting how the Triton changes pH. At a pH above 8 it doesn't really work.
This is my protocol:
3.7% Formaldehyde + 0.1% Triton-X in 1X PBS..
Incubation period = 20mins at 4 Celcius
I also use the BD cytofix and cytoperm and follows its protocol..
I have lost a significant number of cells when i use my own protocol but I can get sufficient cell numbers if I used BD cytofix and cytoperm..
the centrifugation force = 2200rpm for 5mins at 4 Celcius..
I have no idea why 3.7% formaldehyde + 0.1% triton-X can cause significant cell loss..
should I increase the centrifuge force? Please help. thank you
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